Chloroplast transit peptides and methods of their use

ABSTRACT

Methods and compositions are provided for targeting a polypeptide of interest to a chloroplast. Recombinant polynucleotides comprising a nucleotide sequence encoding a chimeric chloroplast transit peptide (CTP) operably linked to a heterologous polynucleotide of interest are provided. In specific embodiments, the chimeric CTP comprises an N-terminal domain operably linked to a central domain operably linked to a C-terminal domain of a CTP to form a chimeric chloroplast transit peptide having CTP activity. Recombinant polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the recombinant polynucleotides. Methods of use of the various sequences are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/488,952, filed May 23, 2011, which is hereby incorporated herein inits entirety by reference.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted Sep. 30, 2013 as a text file named36446_(—)0050U1_Revised_Sequence_Listing.txt, created on Sep. 30, 3013,and having a size of 43,924 bytes is hereby incorporated by referencepursuant to 37 C.F.R. 1.52(e)(5).

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to targeting sequences of interest to achloroplast by employing novel chloroplast transit peptides.

BACKGROUND OF THE INVENTION

Plastids are a heterogeneous family of organelles found ubiquitously inplants and algal cells. Most prominent are the chloroplasts, which carryout such essential processes as photosynthesis and the biosynthesis offatty acids as well as of amino acids. Chloroplasts are complexorganelles composed of six distinct suborganellar compartments: threedifferent membranes (the two envelope membranes and the internalthylakoid membranes) and three compartments (the innermembrane space ofthe envelope, the stroma and the thylakoid lumen). More than 98% of allplastid proteins are translated on cytosolic ribosomes. Such proteinsare posttranslationally targeted to and imported into the organelle. Fora review, see, Jarvis et al. (2008) New Phytologist 179:257-285. Suchtranslocation is mediated by multiprotein complexes in the outer andinner envelope membranes called TOC (Translocon at the Outer envelopemembrane of Chloroplasts) and TIC (Translocon at the Inner envelopemembrane of Chloroplasts). See, Soll et al. (2004) Nature Reviews.Molecular Cell Biology 5:198-208, Bedard et al. (2005) Journal ofExperimental Botany 56:2287-2320, Kessler et al. (2006) Traffic7:248-257, and Smith et al. (2006) Canadian Journal of Botany84:531-542. Once the chloroplast precursor enters the stroma, thetransit peptide is cleaved off, leaving the remaining part of theprotein to take on its final conformation or engage one of a number ofdifferent sorting pathways. See, Keegstra et al. (1999) Plant Cell11:557-570, Jarvis et al. (2004) and Gutensohn et al. (2006) Journal ofPlant Physiology 163:333-347.

Methods and compositions are needed to allow heterologous polypeptidesto be targeted to the chloroplast.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided for targeting a polypeptide ofinterest to a chloroplast. Recombinant polynucleotides comprising anucleotide sequence encoding a chimeric chloroplast transit peptide(CTP) operably linked to a heterologous polynucleotide of interest areprovided. In specific embodiments, the chimeric CTP comprises anN-terminal domain operably linked to a central domain operably linked toa C-terminal domain of a CTP to form a chimeric chloroplast transitpeptide having CTP activity. Recombinant polypeptides encoding the same,as well as, cells, plant cells, plants and seeds are further providedwhich comprise the recombinant polynucleotides. Methods of use of thevarious sequences are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a strategy for developing the recombinant chloroplasttransit peptides provided herein. The origin of each segment of the CTPframework for the recombinant chloroplast transit peptides is provided.

FIG. 2 provides an amino acid alignment of chloroplast transit peptidesfrom various monocot plants. The most frequent amino acids arehighlighted. Starting from the top, the sequences are identified as SEQID NO:13, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:21, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:22, SEQ ID NO:50, SEQ IDNO:18, SEQ ID NO:51, SEQ ID NO:16, SEQ ID NO:14, SEQ ID NO:52, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:15, SEQ IDNO:20, SEQ ID NO:55, SEQ ID NO:23, SEQ ID NO:56, SEQ ID NO:11.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Compositions

A. Overview

In the production of transgenic plants it is often useful to directforeign proteins to specific subcellular locations, e.g., the plastid,vacuole, mitochondria, or ER. When the gene is translated, the resultingprotein has the transit peptide fused to the amino terminus of theprotein of interest, and thus the protein is directed to the desiredsubcellular compartment. Of particular interest is the identification oftransit peptides that will direct transport to a plastid. As usedherein, a “plastid” refers to an organelle present in plant cells thatstores and manufactures chemical compounds used by the cell, such asstarch, fatty acids, terpenes, and that has been derived from aproplastid. Thus, plastids of plants typically have the same geneticcontent. Plastids include chloroplasts, which are responsible forphotosynthesis, amyloplasts, chromoplasts, statoliths, leucoplasts,elaioplasts, and proteinoplasts. Plastids contain photosyntheticmachinery and many additional biosynthetic enzymes including thoseleading to the production of fatty acids, amino acids, carotenoids,terpenoids, and starch. Thus, there is a need for the ability to targetpolypeptides of interest to plastids to modulate or alter thephysiological processes that occur within these organelles. In addition,some polypeptides are toxic when expressed recombinantly in thecytoplasm. Because plastids are subcompartments, it is possible totarget polypeptides of interest to the plastids to sequester them fromthe cytoplasm, and thus allow for higher expression levels. Furthermore,expression of recombinant polypeptides in plastids may facilitateisolation of the polypeptide for various applications. As discussed infurther detail herein, novel chimeric chloroplast transit peptides areprovided which can be used in plastid targeting.

The compositions provided herein include recombinant polynucleotidescomprising a nucleotide sequence encoding a novel chloroplast transitpeptide (CTP) operably linked to a nucleotide sequence encoding apolypeptide of interest. The CTP-encoding sequences disclosed herein,when assembled within a DNA construct such that the CTP-encodingsequence is operably linked to a nucleotide sequence encoding thepolypeptide of interest, facilitate co-translational orpost-translational transport of the peptide of interest to thechloroplast of a plant cell.

B. Chloroplast Transit Peptides

Chloroplasts are organelles found in plant cells and eukaryotic algaethat conduct photosynthesis. The chloroplast is a complex cellularorganelle composed of three membranes: the inner envelope membrane, theouter envelope membrane, and the thylakoid membrane. The membranestogether enclose three aqueous compartments termed the intermediatespace, the stroma, and the thylakoid lumen. While chloroplasts containtheir own circular genome, many constituent chloroplast proteins areencoded by the nuclear genes and are cytoplasmically-synthesized asprecursor forms which contain N-terminal extensions known as chloroplasttransit peptides (CTPs). As used herein, the term “chloroplast transitpeptide” or “CTP” refers to the N-terminal portion of a chloroplastprecursor protein and influences the recognition of the chloroplastsurface and mediates the post-translational translocation ofpre-proteins across the chloroplast envelope and into the varioussubcompartments within the chloroplast (e.g. stroma, thylakoid andthylakoid membrane). Thus, as used herein, a polypeptide having “CTPactivity” comprises a polypeptide which when operably linked to theN-terminal region of a protein of interest facilitates translocation ofthe polypeptide of interest to the chloroplast.

Assays to determine the efficiency by which the CTP sequences providedherein target a protein of interest to a chloroplast are known. See, forexample, Mishkind et al. (1985) J. of Cell. Biol. 100:226-234, which isherein incorporated by reference in its entirety. A reporter gene suchas glucuronidase (GUS), chloramphenicol acetyl transferase (CAT), orgreen fluorescent protein (GFP) is operably linked to the CTP sequence.This fusion is placed behind the control of a suitable promoter, ligatedinto a transformation vector, and transformed into a plant or plantcell. Following an adequate period of time for expression andlocalization into the chloroplast, the chloroplast fraction is extractedand reporter activity assayed. The ability of the CTP sequences totarget and deliver the reporter protein to the chloroplast can becompared to other known CTP sequences. See, de Castro Silva Filho et al.(1996) Plant Mol. Biol. 30: 769-780. Protein import can also be verifiedin vitro through the addition of proteases to the isolated chloroplastfraction. Proteins which were successfully imported into the chloroplastare resistant to the externally added proteases whereas proteins thatremain in the cytosol are susceptible to digestion. Protein import canalso be verified by the presence of functional protein in thechloroplast using standard molecular techniques for detection, byevaluating the phenotype resulting from expression of a chloroplasttargeted protein, or by microscopy.

a. Chimeric Chloroplast Transit Peptides

Recombinant polynucleotides encoding a chimeric CTP operably linked to aheterologous polynucleotide of interest are provided herein. Thechimeric CTPs comprise heterologous domains of known or predicted CTPswhich, when operably linked, have CTP activity.

CTPs have a preference for hydroxylated amino acids (S, T, P) and lackacidic residues. They share a common structural framework comprising anuncharged N-terminal region (“N-terminal domain”), a central region(“central domain”), and a basic arginine-rich amphipathic C-terminalregion (“C-terminal domain”). The domain framework structure of CTPs isprovided in FIG. 1. Thus, the CTPs provided herein comprise 3 domains,an N-terminal domain, a central domain and a C-terminal domain.

As used herein, “N-terminal domain” refers to an N-terminal hydrophobicregion of a CTP comprising uncharged amino acids. The N-terminal domaincan comprise at least 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17,5-18, 5-19, 5-20 or more amino acids from the N-terminus of a CTPsequence generally beginning with MA and ending in G/P. The N-terminaldomain can comprise additional sequences, such as linker sequences, suchthat when operably linked to a central domain and C-terminal domainreconstitutes a CTP having CTP activity.

A “central domain” as used herein, refers to a central region of a CTPcomprising an amino acid sequence lacking acidic amino acids andenriched in serine, threonine, lysine and arginine. The central domaincan comprise at least 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13,4-14, 4-15 or more amino acids from the central region of a CTPsequence. The central domain can also comprise additional sequences suchas linker sequences, such that when operably linked to an N-terminaldomain and a C-terminal domain reconstitutes a CTP having CTP activity.

As used herein, “C-terminal domain” refers to a C-terminal region of aCTP comprising an amino acid sequence which is basic, arginine-rich andpredicted to form an amphiphilic beta strand. The C-terminal domain cancomprise at least 5-10, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22,5-23, 5-24, 5-25, 5-26, 5-27, 5-28, 5-29, 5-30 or more amino acids fromthe C-terminal region of a CTP sequence. The C-terminal domain cancomprise additional sequences such as linker sequences, such that whenoperably linked to an N-terminal domain and a central domainreconstitutes a CTP having CTP activity.

Non-limiting examples of domains for various CTPs are set forth in SEQID NOS: 24-43 and summarized in Table 4.

A “chimeric CTP” provided herein comprises an N-terminal domain, acentral domain, and a C-terminal domain from any CTP in which thesequence of at least one of the domains is heterologous to the sequenceof the other domains and whereby the domains, when operably linked,reconstitute a CTP with CTP activity. As used herein the term “chimeric”refers to a sequence having two or more heterologous sequences linkedtogether. As used herein, “heterologous” in reference to a sequence is asequence that originates from a foreign species, for example, from adifferent CTP, or, if from the same species, is substantially modifiedfrom its native form in composition and/or genomic locus by deliberatehuman intervention. For example, a heterologous domain is intended atleast one of the CTP domains is not from the same CTP, but could be froma different CTP of the same plant species or a different plant species.The chimeric CTPs provided herein can vary in length from about 30, 35,40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65or more amino acid residues in length such that it comprises anN-terminal domain, a central domain, and a C-terminal domain and retainsCTP activity.

The domains of the chimeric CTPs can be from any known or predicted CTPsequence. For example, in some embodiments, the chimeric CTP cancomprise, but is not limited to, an N-terminal domain, a central domainor a C-terminal domain from a CTP from Oryza sativa 1-deoxy-Dxyulose-5-Phosphate Synthase, Oryza sativa-Superoxide dismutase, Oryzasativa-soluble starch synthase, Oryza sativa-NADP-dependent Malic acidenzyme, Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2,Oryza sativa-L-Ascorbate peroxidase 5, Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type or active variants thereof.

In specific, non-limiting, embodiments, the N-terminal domain of thechimeric CTP is from a CTP from Oryza sativa 1-deoxy-Dxyulose-5-Phosphate Synthase, Oryza sativa-NADP-dependent Malic acidenzyme, Zea Mays-Malate dehydrogenase or active variants thereof. Inother non-limiting embodiments, the central domain of the chimeric CTPis from a CTP from Oryza sativa-Superoxide dismutase, Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2, Oryzasativa-L-Ascorbate peroxidase 5, Zea Mays ssRUBISCO or active variantsthereof. In yet other non-limiting embodiments, the C-terminal domain ofthe chimeric CTP is from a CTP from Oryza sativa-soluble starchsynthase, Oryza sativa-Superoxide dismutase, Oryza sativa-Phosphoglucanwater dikinase, Zea Mays Thioredoxin M-type, Zea Mays-beta-glucosidaseor active variants thereof. Non-limiting examples of various CTPs,N-terminal domains, central domains and C-terminal domains of CTPs areset forth in SEQ ID NOS: 13-43.

In one specific embodiment, the chimeric CTP comprises the N-terminaldomain from the Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase CTPor an active variant thereof, the central domain from the Zea MaysssRUBISCO CTP or an active variant thereof, and the C-terminal domain ofthe Zea Mays-beta-glucosidase CTP or an active variant thereof. Inanother specific embodiment, the chimeric CTP comprises the N-terminaldomain from the Zea Mays-Malate dehydrogenase CTP or an active variantthereof, the central domain from the Oryza sativa-Superoxide dismutaseCTP or an active variant thereof, and the C-terminal domain from theOryza sativa-soluble starch synthase CTP or an active variant thereof.In yet another specific embodiment, the chimeric CTP comprises theN-terminal domain from the Oryza sativa-NADP-dependent Malic acid enzymeCTP or active variant thereof, the central domain from the Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 CTP or an activevariant thereof, and the C-terminal domain from the Zea Mays ThioredoxinM-type CTP or an active variant thereof.

Examples of chimeric CTPs are set forth in the amino acid sequences ofSEQ ID NO: 1 (msCTP1) or an active variant or fragment thereof, SEQ IDNO: 2 (msCTP2) or an active variant or fragment thereof and SEQ ID NO: 3(msCTP3) or an active variant or fragment thereof. The domain structuresof the various CTPs provided herein are depicted in FIG. 1.

The chimeric CTPs provided herein can also comprise chimeric domains. Asused herein, a “chimeric domain” refers to an N-terminal domain, centraldomain, or C-terminal domain of a CTP which comprises portions of two ormore heterologous N-terminal domain, central domain, or C-terminaldomain sequences fused together to reconstitute a complete domain. Forexample, a chimeric domain (i.e. a “chimeric N-terminal domain”,“chimeric central domain” or “chimeric C-terminal domain”) providedherein can comprise at least 2, 3, 4 or more heterologous CTP sequencesfused together such that the chimeric domain, when incorporated in achimeric CTP, has CTP activity.

In some embodiments, the chimeric CTPs can comprise at least 1, 2 or 3chimeric domains. In specific embodiments, at least one portion of thechimeric N-terminal domain is from the N-terminal domain of the Oryzasativa-NADP-dependent Malic CTP, Zea Mays-Malate dehydrogenase CTP oractive variants thereof. In other embodiments at least one portion ofthe chimeric central domain is from the central domain of the Oryzasativa-NADP-dependent Malic CTP, Zea Mays-Malate dehydrogenase CTP oractive variants thereof. In yet other embodiments, at least one portionof the chimeric C-terminal domain is from the C-terminal domain of theOryza sativa-soluble starch synthase CTP, Zea Mays Thioredoxin M-typeCTP, Oryza sativa-Superoxide dismutase CTP, Oryza sativa-Phosphoglucanwater dikinase CTP or active variants thereof.

In a specific embodiment, the chimeric CTP comprises a chimericN-terminal domain comprising a portion of the N-terminal domain from theZea Mays-Malate dehydrogenase CTP fused in frame to a portion of theN-terminal domain of the Oryza sativa-NADP-dependent Malic acid enzymeCTP, a central domain from the Zea Mays ssRUBISCO CTP, and a chimericC-terminal domain comprising a portion of the C-terminal domain from theOryza sativa-soluble starch synthase CTP fused in frame to a portion ofthe C-terminal domain from the Zea Mays Thioredoxin M-type CTP, whereinthe chimeric CTP has CTP activity.

In another specific embodiment, the chimeric CTP comprises a chimericN-terminal domain comprising a portion of the Zea Mays-Malatedehydrogenase CTP fused in frame to a portion of the Oryzasativa-NADP-dependent Malic acid enzyme CTP, a chimeric central domaincomprising a portion of the Oryza sativa-L-Ascorbate peroxidase 5 CTPfused in frame to a portion of the Zea Mays ssRUBISCO CTP, and achimeric C-terminal domain comprising a portion of the Oryzasativa-Superoxide dismutase CTP fused in frame to a portion of the Oryzasativa-Phosphoglucan water dikinase CTP, wherein the chimeric CTP hasCTP activity.

Exemplary CTPs comprising chimeric domains are set forth in the aminoacid sequences of SEQ ID NO:4 (msCTP4) or an active or fragment variantthereof and SEQ ID NO:5 (msCTP5) or an active variant or fragmentthereof. Examples of chimeric CTP domain structures are provided in FIG.1.

b. Consensus Chloroplast Transit Peptides

While the chimeric CTPs described in the previous section employed adomain approach for CTP design, it is recognized that other approachescan be used to design chloroplast transit peptides having CTP activity.Provided herein are recombinant polynucleotides encoding CTPs withsequences based on the alignment of various known monocot CTP sequencesand the most frequent amino acids at each position operably linked to aheterologous polynucleotide encoding a polypeptide of interest. FIG. 2provides the alignment of the various monocot CTP sequences used todetermine the most frequent amino acids. The various CTPs were alignedbased on the structural framework of the different domains as describedelsewhere herein and a consensus CTP sequence is provided.

In one embodiment, a CTP is provided comprising the following CTPconsensus sequence:

(SEQ ID NO: 11) MXXXXVXXAAAXXXXSXPXXRXXXGXXXXXXXXXXXXXXXXXAAXX RXXXX::or an active variant thereof, where the X indicates any amino acid.

Based on the consensus sequence, various CTPs can be constructed suchthat the resulting CTP has CTP activity. In some cases, a dominant aminoacid residue may not be apparent. In these cases, one of the morefrequent amino acid residues can be chosen to be incorporated into thesequence. It is recognized that many CTP sequences can be provided fromthe consensus sequence disclosed herein.

In one non-limiting embodiment, a CTP is provided having the followingsequence:

(SEQ ID NO: 6) M ALAS V MA AAA ASVV S F P AG R GSG G SSVLRSRALSLAGSRRSAA AV R R LAL:: (msCTP6)or an active variant or fragment thereof. In another non-limitingembodiment, a CTP sequence is provided having the following sequence:

(SEQ ID NO: 7) M AVAT V LA AAA LAAV S P P GL R SSL G FPVVRRSLPSAARGGSPAA TR R CR AA:: (msCTP7)or an active variant or fragment thereof.

c. Other Components of the CTPs Provided Herein

It is recognized that the various CTPs disclosed herein can be modifiedto improve and/or alter the translocation of the polypeptide of interestinto the chloroplast. For example, the CTP can contain additionalregions that alter or improve the interactions with cytosolic factorsthat facilitate the passage of precursors from the ribosomes to thechloroplast surface. See, for example, Hiltbrunner et al. (2001) Journalof Cell Biology 154:309-316, Jackson-Constan et al. (2001) Biochimica etBiophysica Acta 1541:102-113, both of which are herein incorporated byreference. Other regions can be employed to increase the efficiency ofchloroplast import. See, for example, May et al. (2000) Plant Cell12:53-64, Qbadou et al. (2006) EMBO Journal 25:1837-1837 and Sohrt etal. (2000) Journal of Cell Biology 148:1213-1221, herein incorporated byreference. Such regions may be native (derived from a region of the samechloroplast targeted polypeptide as the CTP) or heterologous to theoperably linked CTP provided herein.

The various CTPs disclosed herein can further comprise additionalsequences which modulate the final location of the polypeptide ofinterest in the chloroplast. For example, the various CTPs disclosedherein could further comprise a thylakoid lumen targeting domain.Proteins to be targeted to the thylakoid lumen bear an additionalcleavable targeting signal, which like the transit peptide, is removedonce translocation is complete. The luminal targeting peptides areextremely similar to the signal peptides that mediate inner membranetransport in bacteria. See, for example, Keegstra et al. (1999) PlantCell 11:557-570, Jarvis (2004) Current Biology 14: R1064-R1077,Gutensohn et al. (2006) Journal of Plant Physiology 163:333-347, andJarvis (2008) New Phytologist 179:257-285, all of which are incorporatedby reference in their entirety, which discuss the various sortingpathways in a chloroplast. Such regions which modulate the location ofthe polypeptide of interest in a chloroplast may be native (derived froma region of the same chloroplast targeted polypeptide as the CTP) orheterologous to the operably linked CTP provided herein.

The term “chloroplast transit peptide cleavage site” refers to a sitebetween two amino acids in a chloroplast-targeting sequence at which thechloroplast processing protease acts. CTPs target the desired protein tothe chloroplast and can facilitate the protein's translocation into theorganelle. This is accompanied by the cleavage of the transit peptidefrom the mature polypeptide or protein at the appropriate transitpeptide cleavage site by a chloroplast processing protease. Accordingly,a CTP can further comprise a suitable cleavage site for the correctprocessing of the pre-protein to the mature polypeptide contained withinthe chloroplast. In one non-limiting example, the CTP cleavage site iswithin the N-terminus of the IP2-127 protein between amino acid 15 and16 in SEQ ID NO: 12, when msCTP4 was used in combination with IP2-127.As discussed above, the sequences beyond the cleaved fragments may beimportant for localization/transport efficiency and be employed with anyof the CTPs disclosed herein.

d. Polynucleotide and Polypeptide Fragments and Variants of CTPs

Fragments and variants of the CTP-sequences (i.e. SEQ ID NOS: 1-7 and13-23) are also encompassed herein. By “fragment” is intended a portionof the polynucleotide or a portion of the amino acid sequence and henceprotein encoded thereby. Fragments of a polynucleotide may encodeprotein fragments that retain CTP activity when reconstituted in a CTPand are thus capable of facilitating the translocation of a polypeptideof interest into the chloroplast of a plant. Alternatively, fragments ofa polynucleotide that are useful as a hybridization probe generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 10, 20,30, 40, 50, 60, 70, 80 nucleotides or up to the full length CTP.

A fragment of a polynucleotide that encodes a biologically activeportion of a CTP-polypeptide will encode at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60 contiguous amino acids, or up tothe total number of amino acids present in any one of SEQ ID NOS: 1, 2,3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or of thevarious chimeric CTPs disclosed herein. Fragments of a CTP-encodingsequence that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of a CTP.

“Variant” CTP is intended to mean a protein derived from the CTP (i.e.SEQ ID NOS: 1-7 and 13-23) by deletion (i.e., truncation at the 5′and/or 3′ end) and/or a deletion or addition of one or more amino acidsat one or more internal sites in the CTP and/or substitution of one ormore amino acids at one or more sites in the CTP, and/or substitution ofone or more of the N-terminal, central, or C-terminal domains of the CTPand/or substitution of a portion of one or more of the N-terminal,central, or C-terminal domains of the CTP. Variant proteins encompassedare biologically active, that is they continue to possess the desiredbiological activity of the CTP, that is, have CTP activity whenreconstituted in a CTP. Such variants may result from, for example,genetic polymorphism or from human manipulation.

For polynucleotides encoding a CTP, a variant comprises a polynucleotidehaving a deletion (i.e., truncations) at the 5′ and/or 3′ end and/or adeletion and/or addition of one or more nucleotides at one or moreinternal sites within the polynucleotide and/or a substitution of one ormore nucleotides at one or more sites in the polynucleotide and/orsubstitution of one or more of the N-terminal, central, or C-terminaldomains of the polynucleotide encoding the CTP and/or substitution of aportion of one or more of the N-terminal, central, or C-terminal domainsof the polynucleotide encoding the CTP. Variant polynucleotides alsoinclude synthetically derived polynucleotides, such as those generated,for example, by using site-directed mutagenesis or gene synthesis butwhich still encode a CTP.

Biologically active variants of a CTP provided herein (and thepolynucleotide encoding the same) will have at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the polypeptide of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or to any N-terminaldomain or portion thereof, any central domain or portion thereof or anyC-terminal domain or portion thereof from any one of SEQ ID NOS: 1-7,13-43 or any of the other CTPs disclosed herein.

The CTP-sequences and the active variants and fragments thereof may bealtered in various ways including amino acid substitutions, deletions,truncations, and insertions. Methods for such manipulations aregenerally known in the art. For example, amino acid sequence variantsand fragments of the CTPs can be prepared by mutations in the DNA.Methods for mutagenesis and polynucleotide alterations are well known inthe art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and optimallywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more differentCTP-sequences can be manipulated to create a new CTP possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between the CTP sequences disclosedherein and other known CTPs to obtain a new polynucleotide coding for apolypeptide with an improved property of interest, such as an improvedefficiency of transport to the chloroplast. Strategies for such DNAshuffling are known in the art. See, for example, Stemmer (1994) Proc.Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391;Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J.Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

e. Sequence Comparisons

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percent sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene sequenceor protein sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polypeptide sequence, wherein the polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two polypeptides.Generally, the comparison window is at least 5, 10, 15, or 20 contiguousamino acids in length, or it can be 30, 40, 50, 100, or longer. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polypeptide sequencea gap penalty is typically introduced and is subtracted from the numberof matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. BLASTP protein searches can beperformed using default parameters. See,blast.ncbi.nlm.nih.gov/Blast.cgi.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, or PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTP forproteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

In one embodiment, sequence identity/similarity values provided hereinrefer to the value obtained using GAP Version 10 using the followingparameters: % identity and % similarity for an amino acid sequence usingGAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix;or any equivalent program thereof. By “equivalent program” is intendedany sequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity). When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percent sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percent sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentsequence identity.

(e) Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacids substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols such as Gapped BLAST 2.0. The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each additional empty amino acid position inserted into analready opened gap. The gap existence penalty is imposed for theintroduction of a single amino acid gap in one of the aligned sequences,and the gap extension penalty is imposed for each additional empty aminoacid position inserted into an already opened gap. The alignment isdefined by the amino acids positions of each sequence at which thealignment begins and ends, and optionally by the insertion of a gap ormultiple gaps in one or both sequences, so as to arrive at the highestpossible score. While optimal alignment and scoring can be accomplishedmanually, the process is facilitated by the use of acomputer-implemented alignment algorithm, e.g., gapped BLAST 2.0,described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, andmade available to the public at the National Center for BiotechnologyInformation Website (http://www.ncbi.nlm.nih.gov). Optimal alignments,including multiple alignments, can be prepared using, e.g., PSI-BLAST,available through http://www.ncbi.nlm.nih.gov and described by Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402.

As used herein, similarity score and bit score is determined employingthe BLAST alignment used the BLOSUM62 substitution matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. For the samepair of sequences, if there is a numerical difference between the scoresobtained when using one or the other sequence as query sequences, agreater value of similarity score is selected.

C. Polynucleotides/Polypeptides of Interest

Any heterologous polynucleotide of interest (i.e., the “polypeptide ofinterest”) may be used with the CTP-encoding sequences disclosed herein(i.e. the various chimeric CTPs disclosed herein and/or SEQ ID NOS: 1,2, 3, 4, 5, 6, 7 or active variants or fragments thereof). It isrecognized that any polypeptides of interest can be operably linked tothe CTP-encoding sequences provided herein and expressed in a plant.

Such polynucleotides/polypeptides of interest include, but are notlimited to, herbicide-tolerance coding sequences, insecticidal codingsequences, nematicidal coding sequences, antimicrobial coding sequences,antifungal coding sequences, antiviral coding sequences, abiotic andbiotic stress tolerance coding sequences, or sequences modifying planttraits such as yield, grain quality, nutrient content, starch qualityand quantity, nitrogen fixation and/or utilization, and oil contentand/or composition. More specific polynucleotides of interest include,but are not limited to, genes that improve crop yield, polypeptides thatimprove desirability of crops, genes encoding proteins conferringresistance to abiotic stress, such as drought, nitrogen, temperature,salinity, toxic metals or trace elements, or those conferring resistanceto toxins such as pesticides and herbicides, or to biotic stress, suchas attacks by fungi, viruses, bacteria, insects, and nematodes, anddevelopment of diseases associated with these organisms.

An “herbicide resistance protein” or a protein resulting from expressionof an “herbicide resistance-encoding nucleic acid molecule” includesproteins that confer upon a cell the ability to tolerate a higherconcentration of an herbicide than cells that do not express theprotein, or to tolerate a certain concentration of an herbicide for alonger period of time than cells that do not express the protein.Herbicide resistance traits may be introduced into plants by genescoding for resistance to herbicides that act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides, genes coding for resistance to herbicides that act toinhibit the action of glutamine synthase, such as phosphinothricin orbasta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene andthe GAT gene), HPPD inhibitors (e.g., the HPPD gene) or other such genesknown in the art. See, for example, U.S. Pat. Nos. 7,626,077, 5,310,667,5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and U.S.Provisional Application No. 61/401,456, each of which is hereinincorporated by reference.

Polynucleotides that improve crop yield include dwarfing genes, such asRht1 and Rht2 (Peng et al. (1999) Nature 400:256-261), and those thatincrease plant growth, such as ammonium-inducible glutamatedehydrogenase. Polynucleotides that improve desirability of cropsinclude, for example, those that allow plants to have a reducedsaturated fat content, those that boost the nutritional value of plants,and those that increase grain protein. Polynucleotides that improve salttolerance are those that increase or allow plant growth in anenvironment of higher salinity than the native environment of the plantinto which the salt-tolerant gene(s) has been introduced.

Polynucleotides/polypeptides that influence amino acid biosynthesisinclude, for example, anthranilate synthase (AS; EC 4.1.3.27) whichcatalyzes the first reaction branching from the aromatic amino acidpathway to the biosynthesis of tryptophan in plants, fungi, andbacteria. In plants, the chemical processes for the biosynthesis oftryptophan are compartmentalized in the chloroplast. See, for example,US Pub. 20080050506, herein incorporated by reference. Additionalsequences of interest include Chorismate Pyruvate Lyase (CPL) whichrefers to a gene encoding an enzyme which catalyzes the conversion ofchorismate to pyruvate and pHBA. The most well characterized CPL genehas been isolated from E. coli and bears the GenBank accession numberM96268. See, U.S. Pat. No. 7,361,811, herein incorporated by reference.

These polynucleotide sequences of interest may encode proteins involvedin providing disease or pest resistance. By “disease resistance” or“pest resistance” is intended that the plants avoid the harmful symptomsthat are the outcome of the plant-pathogen interactions. Diseaseresistance and insect resistance genes such as lysozymes or cecropinsfor antibacterial protection, or proteins such as defensins, glucanasesor chitinases for antifungal protection, or Bacillus thuringiensisendotoxins, protease inhibitors, collagenases, lectins, or glycosidasesfor controlling nematodes or insects are all examples of useful geneproducts.

In some embodiments, a CTP provided herein is operably linked to aheterologous polypeptide of interest comprising an insecticidal proteinand expression of the polypeptide controls a pest (i.e. insecticidalactivity). As used herein, by “controlling a pest” or “controls a pest”is intended any affect on a pest that results in limiting the damagethat the pest causes. Controlling a pest includes, but is not limitedto, killing the pest, inhibiting development of the pest, alteringfertility or growth of the pest in such a manner that the pest providesless damage to the plant, decreasing the number of offspring produced,producing less fit pests, producing pests more susceptible to predatorattack, or deterring the pests from eating the plant.

“Pest” includes, but is not limited to, insects, fungi, bacteria,viruses, nematodes, mites, ticks, and the like. Insect pests includeinsects selected from the orders Coleoptera, Diptera, Hymenoptera,Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,etc., particularly Coleoptera, Lepidoptera, and Diptera. Viruses includebut are not limited to tobacco or cucumber mosaic virus, ringspot virus,necrosis virus, maize dwarf mosaic virus, etc. Nematodes include but arenot limited to parasitic nematodes such as root knot, cyst, and lesionnematodes, including Heterodera spp., Meloidogyne spp., and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cystnematode); and Globodera rostochiensis and Globodera pailida (potatocyst nematodes). Lesion nematodes include but are not limited toPratylenchus spp. Fungal pests include those that cause leaf, yellow,stripe and stem rusts.

In other embodiments, a polypeptide of interest comprises a Bacillusthuringiensis polypeptide having insecticidal activity (i.e. controls apest). Some examples of Bacillus thuringiensis toxic proteins includethe Cry proteins. Other Bacillus thuringiensis toxic proteins aredescribed in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109, herein incorporated byreference. In a specific embodiment, the Bacillus thuringiensispolypeptide is IP2-127 (SEQ ID NO: 12) or an active variant or fragmentthereof. IP2-127 is a Cry2 protein of Bacillus thuringiensis withinsecticidal activity. The IP2-127 protein may be modified to comprise,for example, a short linker sequence or a reporter gene in order toallow detection of the protein. An Example of a modified IP2-127 proteinsequence is set forth in SEQ ID NO: 8 or an active variant or fragmentthereof and is encoded by the polynucleotide sequence set forth in SEQID NO:9 or an active variant or fragment thereof and comprises anIP2-127-AcGFP fusion protein.

It is recognized that any polypeptide of interest may be modified tocomprise, for example, a short linker sequence or a reporter gene inorder to allow detection of the protein in the chloroplast.

Active variants or fragments of polynucleotides/polypeptides of interest(i.e. SEQ ID NO:12) are also provided. Such active variants can compriseat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to the nativepolynucleotide/polypeptide of interest, wherein the active variantsretain biological activity and are functional in chloroplasts. Activefragments can comprise nucleic acid/amino acid sequences having at least20, 25, 30, 35, 40, 50, 60, 70, 80, 100, 150, or more consecutivenucleic acids/amino acids of the native polynucleotide/polypeptide ofinterest, where the active fragments retain biological activity and arefunctional in chloroplasts. As used herein, a “native” polynucleotide orpolypeptide comprises a naturally occurring nucleotide sequence or aminoacid sequence, respectively. Methods to determine sequenceidentity/sequence similarity are described in detail elsewhere herein.

D. Plants

Compositions comprising a cell, a transgenic plant cell, a transgenicplant, transgenic plant parts and seeds, plant explants and grain havingthe recombinant polynucleotide encoding a CTP operably linked to aheterologous polynucleotide encoding a polypeptide of interest arefurther provided. In one embodiment, a cell, a plant cell, a plant,plant parts and seeds, plant explants and grain comprise at least onepolynucleotide encoding a CTP provided herein (i.e. The various chimericCTPs disclosed herein and/or SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or activevariants or fragments thereof) operably linked to a polypeptide ofinterest. The CTP may comprise a chimeric CTP, a chimeric CTP comprisingchimeric domains, or a CTP comprising a consensus sequence as describedin detail elsewhere herein. In some cases, the polynucleotide encodingthe polypeptide of interest can comprise an insecticidal protein thatcontrols a pest, a Bacillus thuringiensis protein having insecticidalactivity, or an IP2-127 protein (i.e. SEQ ID NO:12) or active variant orfragment thereof.

As used herein, the term plant includes whole plants, plant organs,plant tissues, seeds and plant cells and progeny of the same, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers, and the like. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included, provided that these partscomprise the introduced recombinant polynucleotides.

A transformed plant or transformed plant cell provided herein is one inwhich genetic alteration, such as transformation, has been affected asto a gene of interest, or is a plant or plant cell which is descendedfrom a plant or cell so altered and which comprises the alteration. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. Accordingly, a “transgenic plant” is a plantthat contains a transgene, whether the transgene was introduced intothat particular plant by transformation or by breeding; thus,descendants of an originally-transformed plant are encompassed by thedefinition. A “subject plant or plant cell” is one in which geneticalteration, such as transformation, has been affected as to a gene ofinterest, or is a plant or plant cell which is descended from a plant orcell so altered and which comprises the alteration. A “control” or“control plant” or “control plant cell” provides a reference point formeasuring changes in phenotype of the subject plant or plant cell. Acontrol plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich does not express the CTP operably linked to a polypeptide ofinterest, such as a construct comprising a marker gene); (c) a plant orplant cell which is a non-transformed segregant among progeny of asubject plant or plant cell; (d) a plant or plant cell geneticallyidentical to the subject plant or plant cell but which is not exposed toconditions or stimuli that would induce expression of the recombinantpolynucleotide; or (e) the subject plant or plant cell itself, underconditions in which the recombinant polynucleotide is not expressed.

Plant cells that have been transformed to have a recombinantpolynucleotide encoding a CTP operably linked to a polypeptide ofinterest provided herein can be grown into whole plants. Theregeneration, development, and cultivation of plants from single plantprotoplast transformants or from various transformed explants is wellknown in the art. See, for example, McCormick et al. (1986) Plant CellReports 5:81-84; Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988). This regeneration and growth process typically includes thesteps of selection of transformed cells, culturing those individualizedcells through the usual stages of embryonic development through therooted plantlet stage. Transgenic embryos and seeds are similarlyregenerated. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil. Preferably,the regenerated plants are self-pollinated to provide homozygoustransgenic plants. Otherwise, pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important lines.Conversely, pollen from plants of these important lines is used topollinate regenerated plants. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the compositions presented herein provide transformedseed (also referred to as “transgenic seed”) having a polynucleotideprovided herein, for example, a recombinant polynucleotide encoding aCTP operably linked to a polypeptide of interest, stably incorporatedinto their genome.

The recombinant polynucleotides disclosed herein may be used fortransformation of any plant species, including, but not limited to,monocots (e.g., maize, sugarcane, wheat, rice, barley, sorghum, or rye)and dicots (e.g., soybean, Brassica, sunflower, cotton, or alfalfa).Examples of plant species of interest include, but are not limited to,corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include, but not limited to, tomatoes (Lycopersiconesculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), andmembers of the genus Cucumis such as cucumber (C. sativus), cantaloupe(C. cantalupensis), and musk melon (C. melo). Ornamentals include, butnot limited to, azalea (Rhododendron spp.), hydrangea (Macrophyllahydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips(Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima),and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specificembodiments, plants of the present invention are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments,corn and soybean plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

In some embodiments, the recombinant polynucleotides comprising theCTP-encoding sequence operably linked to the polynucleotide encoding thepolypeptide of interest are engineered into a molecular stack. Thus, thevarious plants, plant cells and seeds disclosed herein can furthercomprise one or more traits of interest, and in more specificembodiments, the plant, plant part or plant cell is stacked with anycombination of polynucleotide sequences of interest in order to createplants with a desired combination of traits. As used herein, the term“stacked” includes having the multiple traits present in the same plant.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology, orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes (trans) or contained onthe same transformation cassette (cis). Expression of the sequences canbe driven by the same promoter or by different promoters. In certaincases, it may be desirable to introduce a transformation cassette thatwill suppress the expression of the polynucleotide of interest. This maybe combined with any combination of other suppression cassettes oroverexpression cassettes to generate the desired combination of traitsin the plant. It is further recognized that polynucleotide sequences canbe stacked at a desired genomic location using a site-specificrecombination system. See, for example, WO99/25821, WO99/25854,WO99/25840, WO99/25855, and WO99/25853, all of which are hereinincorporated by reference.

Depending on the polypeptide of interest, the transgenic plants, plantcells or seeds expressing a recombinant polynucleotide provided hereinmay have a change in phenotype, including but not limited to, an alteredpathogen or insect defense mechanism, an increased resistance to one ormore herbicides, an increased ability to withstand stressfulenvironmental conditions, a modified ability to produce starch, amodified level of starch production, a modified oil content and/orcomposition, a modified carbohydrate content and/or composition, amodified ability to utilize, partition and/or store nitrogen, and thelike.

E. Polynucleotide Constructs

Also provided are isolated or recombinant polynucleotides and nucleicacid constructs that encode the CTPs disclosed herein (i.e. the variouschimeric CTPs disclosed herein and/or SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 oractive variants or fragments thereof) operably linked to apolynucleotide encoding a polypeptide of interest. As used herein,“encodes” or “encoding” refers to a DNA sequence which can be processedto generate an RNA and/or polypeptide.

The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acidsequence,” and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof. The use of the term “polynucleotide”is not intended to limit the present invention to polynucleotidescomprising DNA. Those of ordinary skill in the art will recognize thatpolynucleotides, can comprise ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The polynucleotides provided herein also encompass all formsof sequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

The compositions provided herein can comprise an isolated orsubstantially purified polynucleotide. An “isolated” or “purified”polynucleotide is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Optimally, an “isolated” polynucleotide is free ofsequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived.

Further provided are recombinant polynucleotides comprising the CTPsequences and polynucleotide sequences encoding the polypeptides ofinterest. The terms “recombinant polynucleotide” and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial or heterologous combination of nucleic acidsequences, e.g., regulatory and coding sequences that are not foundtogether in nature. For example, a recombinant polynucleotide cancomprise a chimeric CTP operably linked to a heterologous polynucleotideencoding a polypeptide of interest. In other embodiments, a recombinantconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The recombinant polynucleotides disclosed herein can be provided inexpression cassettes for expression in a plant or other organism or celltype of interest. The cassette can include 5′ and 3′ regulatorysequences operably linked to the recombinant polynucleotide or activevariant or fragment thereof. “Operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites and/or recombinationsites for insertion of the recombinant polynucleotide or active variantor fragment thereof to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a CTP-encoding sequence or active variant orfragment thereof operably linked to a polynucleotide encoding apolypeptide of interest and a transcriptional and translationaltermination region (i.e., termination region) functional in plants. Theregulatory regions (i.e., promoters, transcriptional regulatory regions,and translational termination regions) and/or the CTP-encoding sequenceand/or the polynucleotide encoding the polypeptide of interest may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the CTP-encoding sequence and/or thepolynucleotide encoding the polypeptide of interest may be heterologousto the host cell or to each other. In specific embodiments, theCTP-encoding sequence is operably linked to the 5′ end of thepolynucleotide of interest, such that, in the resulting recombinantpolypeptide, the CTP is operably linked to the N-terminal region of thepolypeptide of interest.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, for example, from a differentCTP, or, if from the same species, is substantially modified from itsnative form in composition and/or genomic locus by deliberate humanintervention. For example, a heterologous domain is intended at leastone of the CTP domains is not from the same CTP, but could be from adifferent CTP of the same plant species or a different plant species. Inanother example, a promoter operably linked to a heterologouspolynucleotide is from a species different from the species from whichthe polynucleotide was derived, or, if from the same/analogous species,one or both are substantially modified from their original form and/orgenomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked polynucleotide sequenceof interest, may be native with the plant host, or may be derived fromanother source (i.e., foreign or heterologous) to the promoter, the CTP,the polynucleotide sequence of interest, the plant host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation. Toward this end, adapters or linkers may be employed tojoin the DNA fragments or other manipulations may be involved to providefor convenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al.(1987) Plant Physiol. 84:965-968.

A number of promoters can be used to express the recombinantpolynucleotides provided herein. The promoters can be selected based onthe desired outcome. It is recognized that different applications can beenhanced by the use of different promoters in the expression constructsto modulate the timing, location and/or level of expression of therecombinant polynucleotide. Such expression constructs may also contain,if desired, a promoter regulatory region (e.g. one conferring inducible,constitutive, environmentally- or developmentally regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal. including the nativepromoter of the polynucleotide sequence of interest.

In some embodiments, an expression construct provided herein can becombined with constitutive, tissue-preferred, or other promoters forexpression in plants. Examples of constitutive promoters include, forexample, the cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region, the 1′- or 2′-promoter derived from T-DNA ofAgrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter,the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8promoter and other transcription initiation regions from various plantgenes known to those of skill. If low level expression is desired, weakpromoter(s) may be used. Weak constitutive promoters include, forexample, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S.Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Otherconstitutive promoters include, for example, U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated byreference.

Examples of inducible promoters are the Adh1 promoter which is inducibleby hypoxia or cold stress, the Hsp70 promoter which is inducible by heatstress, the PPDK promoter and the pepcarboxylase promoter which are bothinducible by light. Also useful are promoters which are chemicallyinducible, such as the In2-2 promoter which is safener induced (U.S.Pat. No. 5,364,780), the ERE promoter which is estrogen induced, and theAxig1 promoter which is auxin induced and tapetum specific but alsoactive in callus (PCT US01/22169).

Examples of promoters under developmental control include promoters thatinitiate transcription preferentially in certain tissues, such asleaves, roots, fruit, seeds, or flowers. An exemplary promoter is theanther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051).Examples of seed-preferred promoters include, but are not limited to, 27kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986)Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21):6426; andKloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244. Promotersthat express in the embryo, pericarp, and endosperm are disclosed inU.S. Pat. No. 6,225,529 and PCT publication WO 00/12733. The disclosuresfor each of these are incorporated herein by reference in theirentirety.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionor a recombinant polynucleotide within a particular plant tissue.Tissue-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590. In addition, the promotersof cab and rubisco can also be used. See, for example, Simpson et al.(1958) EMBO J 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglyphosate, glufosinate ammonium, bromoxynil, sulfonylureas, dicamba,and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad Aci.USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be employed herein, including for example,Ac-GFP as described in Examples 2 and 3.

II. Methods of Introducing

The methods provided herein comprise introducing into a cell, plantcell, plant or seed a recombinant polynucleotide or nucleic acidconstruct encoding a CTP provided herein (i.e. Any of the chimeric CTPsprovided herein and/or SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or activevariants or fragments thereof) operably linked to a heterologouspolynucleotide encoding a polypeptide of interest.

In some embodiments, the CTP introduced in the recombinantpolynucleotide can be a chimeric CTP, a chimeric CTP comprising at leastone chimeric domain, or a CTP comprising a consensus sequence asdescribed in detail elsewhere herein. The CTP may be linked to anypolypeptide of interest. For example, the polypeptide of interest cancomprise an insecticidal protein whose expression controls a pest, aBacillus thuringiensis polypeptide having insecticidal activity, or anIP2-127 polypeptide (i.e. SEQ ID NO:12 or an active variant or fragmentthereof).

The methods provided herein do not depend on a particular method forintroducing a sequence into the host cell, only that the polynucleotidegains access to the interior of a least one cell of the host. Methodsfor introducing polynucleotides into host cells (i.e. plants) are knownin the art and include, but are not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

The terms “introducing” and “introduced” are intended to mean providinga nucleic acid (e.g., recombinant polynucleotide) or protein into acell. Introduced includes reference to the incorporation of a nucleicacid into a eukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell, and includes reference to thetransient provision of a nucleic acid or protein to the cell. Introducedincludes reference to stable or transient transformation methods, aswell as sexually crossing. Thus, “introduced” in the context ofinserting a nucleic acid fragment (e.g., a recombinant polynucleotide)into a cell, means “transfection” or “transformation” or “transduction”and includes reference to the incorporation of a nucleic acid fragmentinto a eukaryotic or prokaryotic cell where the nucleic acid fragmentmay be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid, or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA).

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a host (i.e., a plant) integrates into thegenome of the plant and is capable of being inherited by the progenythereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the host (i.e., a plant) and expressedtemporally.

Transformation protocols as well as protocols for introducingpolynucleotide sequences into plants may vary depending on the type ofplant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polynucleotides intoplant cells include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend etal., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, Sanford et al., U.S.Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al.,U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomeset al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the recombinant polynucleotides disclosedherein can be provided to a plant using a variety of transienttransformation methods. Such transient transformation methods include,but are not limited to, the introduction of the recombinantpolynucleotide or variants thereof directly into the plant. Such methodsinclude, for example, microinjection or particle bombardment. See, forexample, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura etal. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad.Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the polynucleotides can be transiently transformed intothe plant using techniques known in the art. Such techniques includeviral vector system and the precipitation of the polynucleotide in amanner that precludes subsequent release of the DNA. Thus, thetranscription from the particle-bound DNA can occur, but the frequencywith which it is released to become integrated into the genome isgreatly reduced. Such methods include the use of particles coated withpolyethylimine (PEI; Sigma #P3143).

In other embodiments, recombinant polynucleotides disclosed herein maybe introduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct provided herein within a viral DNA or RNA molecule.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the recombinant polynucleotides provided herein can be contained in atransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The recombinantpolynucleotide is thereby integrated at a specific chromosomal positionin the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, transformed seed (also referred to as “transgenic seed”)having a recombinant polynucleotide disclosed herein, for example, anexpression cassette provided herein, stably incorporated into theirgenome is provided.

III. Methods of Use

Provided herein is a method of targeting a polypeptide of interest to achloroplast comprising expressing a recombinant polynucleotide encodinga CTP provided herein (i.e. Any of the chimeric CTPs provided hereinand/or SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or active variants or fragmentsthereof) operably linked to a heterologous polynucleotide encoding apolypeptide of interest in a cell, plant cell, plant, plant part orseed.

The methods further provide a CTP comprising a chimeric CTP, a chimericCTP comprising at least one chimeric domain, or a CTP comprising aconsensus sequence as described in detail elsewhere herein. Therecombinant polynucleotide provided in the methods can comprise a CTPprovided herein linked to any polypeptide of interest. For example, thepolypeptide of interest can comprise an insecticidal protein whoseexpression controls a pest, a Bacillus thuringiensis polypeptide havinginsecticidal activity, or an IP2-127 polypeptide (i.e. SEQ ID NO:12 oran active variant or fragment thereof).

Methods of the present invention are directed to the proper expression,translocation, and processing of chloroplast-targeted sequences inplants and plant cells under the control of the CTP sequences disclosedherein. For the purposes of the present invention, a “processed”chloroplast targeted protein is one in which the CTP has been removed.At the time of translocation of a chloroplast targeted protein into thechloroplast of a plant cell, the CTP is removed from the targetedprotein by cleavage at a particular “cleavage site” between the CTP andthe mature protein. The cleavage site can be determined experimentally,or may be predicted based on sequence structure (e.g., by alignment ofthe unprocessed protein with chloroplast targeted proteins in which thecleavage site is known, by analyzing the sequence for the presence ofcharacteristic CTP domains, and the like) or by using one or morealgorithms for cleavage site prediction (e.g., SignalP or PSORT).

Depending on the polypeptide of interest targeted to the chloroplast,the transgenic plants may have a change in phenotype, including, but notlimited to, an altered pathogen or insect defense mechanism, anincreased resistance to one or more herbicides, an increased ability towithstand stressful environmental conditions, a modified ability toproduce starch, a modified level of starch production, a modified oilcontent and/or composition, a modified ability to utilize, partitionand/or store nitrogen, and the like. These results can be achievedthrough the expression and targeting of a polypeptide of interest tochloroplasts in plants, wherein the polypeptide of interest functions inthe chloroplast. The CTP sequences provided herein are useful fortargeting native sequences as well as heterologous (non-native)sequences in plants.

Non-limiting examples of methods and compositions disclosed herein areas follows:

1. A recombinant polynucleotide encoding a chloroplast transit peptide(CTP) operably linked to a heterologous polynucleotide encoding apolypeptide of interest, wherein the CTP comprises

-   -   a) an amino acid sequence comprising the amino acids of SEQ ID        NOS: 6 or 7;    -   b) an amino acid sequence having at least 85% sequence identity        to SEQ ID NOS: 6 or 7, wherein said amino acid sequence has CTP        activity; or,    -   c) an amino acid sequence having at least 17 consecutive amino        acids of SEQ ID NOS: 6 or 7, wherein said amino acid sequence        has CTP activity.

2. A recombinant polynucleotide encoding a chimeric chloroplast transitpeptide (CTP) operably linked to a heterologous polynucleotide encodinga polypeptide of interest, wherein said chimeric CTP comprises anN-terminal domain, a central domain, and a C-terminal domain, or variantthereof, wherein at least one of said N-terminal domain, said centraldomain, said C-terminal domain or variant thereof is heterologous to atleast one of said domains.

3. The recombinant polynucleotide of embodiment 2, wherein saidN-terminal domain, said central domain or said C-terminal domain is froma CTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase, Oryzasativa-Superoxide dismutase, Oryza sativa-soluble starch synthase, Oryzasativa-NADP-dependent Malic acid enzyme, Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2, Oryzasativa-L-Ascorbate peroxidase 5, Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type or active variants thereof.

4. The recombinant polynucleotide of embodiment 2 or 3, wherein saidN-terminal domain is from a CTP from Oryza sativa 1-deoxy-Dxyulose-5-Phosphate Synthase, Oryza sativa-NADP-dependent Malic acidenzyme, Zea Mays-Malate dehydrogenase or active variants thereof.

5. The recombinant polynucleotide of embodiment 2 or 3, wherein saidcentral domain is from a CTP from Oryza sativa-Superoxide dismutase,Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2, Oryzasativa-L-Ascorbate peroxidase 5, Zea Mays ssRUBISCO or active variantsthereof.

6. The recombinant polynucleotide of embodiment 2 or 3, wherein saidC-terminal domain is from a CTP from Oryza sativa-soluble starchsynthase, Oryza sativa-Superoxide dismutase, Oryza sativa-Phosphoglucanwater dikinase, Zea Mays Thioredoxin M-type, Zea Mays-beta-glucosidaseor active variants thereof.

7. The recombinant polynucleotide of embodiment 2 or 3, wherein saidN-terminal domain is from the Oryza sativa 1-deoxy-D xyulose-5-PhosphateSynthase CTP or an active variant thereof, said central domain is fromthe Zea Mays ssRUBISCO CTP or an active variant thereof and saidC-terminal domain is from the Zea Mays-beta-glucosidase CTP or an activevariant thereof.

8. The recombinant polynucleotide of embodiment 2 or 3, wherein saidN-terminal domain is from the Zea Mays-Malate dehydrogenase CTP or anactive variant thereof, said central domain is from the Oryzasativa-Superoxide dismutase CTP or an active variant or thereof and saidC-terminal domain is from the Oryza sativa-soluble starch synthase CTPor an active variant thereof.

9. The recombinant polynucleotide of embodiment 2 or 3, wherein saidN-terminal domain is from the Oryza sativa-NADP-dependent Malic acidenzyme CTP or an active variant thereof, said central domain is from theOryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 CTP or anactive variant thereof and said C-terminal domain is from the Zea MaysThioredoxin M-type CTP or an active variant thereof.

10. The recombinant polynucleotide of embodiment 2, wherein at least oneof said N-terminal domain, said central domain, or said C-terminaldomain comprises a chimeric domain.

11. The recombinant polynucleotide of embodiment 10, wherein at leastone portion of said chimeric N-terminal domain is from the N-terminaldomain of the Oryza sativa-NADP-dependent Malic CTP, Zea Mays-Malatedehydrogenase CTP or active variants thereof.

12. The recombinant polynucleotide of embodiment 10, wherein at leastone portion of said chimeric central domain is from the central domainof the Oryza sativa-L-Ascorbate peroxidase 5 CTP, Zea Mays ssRUBISCO CTPor active variants thereof.

13. The recombinant polynucleotide of embodiment 10, wherein at leastone portion of said chimeric C-terminal domain is from the C-terminaldomain of the Oryza sativa-soluble starch synthase CTP, Zea MaysThioredoxin M-type CTP, Oryza sativa-Superoxide dismutase CTP, Oryzasativa-Phosphoglucan water dikinase CTP or active variants thereof.

14. The recombinant polynucleotide of embodiment 10, wherein saidchimeric CTP comprises

-   -   a) a chimeric N-terminal domain, wherein said chimeric        N-terminal domain comprises a portion of the N-terminal domain        from the Zea Mays-Malate dehydrogenase CTP fused in frame to a        portion of the N-terminal domain of the Oryza        sativa-NADP-dependent Malic acid enzyme CTP;    -   b) a central domain, wherein said central domain is from the Zea        Mays ssRUBISCO CTP; and,    -   c) a chimeric C-terminal domain, wherein said chimeric        C-terminal domain comprises a portion of the C-terminal domain        from the Oryza sativa-soluble starch synthase CTP fused in frame        to a portion of the C-terminal domain from the Zea Mays        Thioredoxin M-type CTP;

wherein said chimeric CTP has CTP activity.

15. The recombinant polynucleotide of embodiment 10, wherein saidchimeric CTP comprises

-   -   a) a chimeric N-terminal domain, wherein said chimeric        N-terminal domain comprises a portion of the N-terminal domain        from the Zea Mays-Malate dehydrogenase CTP fused in frame to a        portion of the N-terminal domain of the Oryza        sativa-NADP-dependent Malic acid enzyme CTP;    -   b) a chimeric central domain, wherein said chimeric central        domain comprises a portion of the central domain from the Oryza        sativa-L-Ascorbate peroxidase 5 CTP fused in frame to a portion        of the central domain of the Zea Mays ssRUBISCO CTP; and,    -   c) a chimeric C-terminal domain, wherein said chimeric        C-terminal domain comprises a portion of the C-terminal domain        from the Oryza sativa-Superoxide dismutase CTP fused in frame to        a portion of the C-terminal domain of the Oryza        sativa-Phosphoglucan water dikinase CTP;

wherein said chimeric CTP has CTP activity.

16. The recombinant polynucleotide of embodiment 3, wherein the chimericCTP comprises

-   -   a) an amino acid sequence comprising the amino acids of SEQ ID        NOS: 1, 2 or 3;    -   b) an amino acid sequence having at least 85% sequence identity        to SEQ ID NOS: 1, 2 or 3, wherein said amino acid sequence has        CTP activity; or    -   c) an amino acid sequence having at least 17 consecutive amino        acids of SEQ ID NOS: 1, 2 or 3, wherein said amino acid sequence        has CTP activity.

17. The recombinant polynucleotide of embodiment 14, wherein thechimeric CTP comprises

-   -   a) an amino acid sequence comprising the amino acids of SEQ ID        NO: 4;    -   b) an amino acid sequence having at least 85% sequence identity        to SEQ ID NO: 4, wherein said amino acid sequence has CTP        activity; or    -   c) an amino acid sequence having at least 17 consecutive amino        acids of SEQ ID NO: 4, wherein said amino acid sequence has CTP        activity.

18. The recombinant polynucleotide of embodiment 15, wherein thechimeric CTP comprises

-   -   a) an amino acid sequence comprising the amino acids of SEQ ID        NO: 5;    -   b) an amino acid sequence having at least 85% sequence identity        to SEQ ID NO: 5, wherein said amino acid sequence has CTP        activity; or    -   c) an amino acid sequence having at least 17 consecutive amino        acids of SEQ ID NO: 5, wherein said amino acid sequence has CTP        activity.

19. The recombinant polynucleotide of any one of embodiments 1-18,wherein said polypeptide of interest comprises a Bacillus thuringiensispolypeptide having insecticidal activity.

20. The recombinant polynucleotide of embodiment 19, wherein saidBacillus thuringiensis polypeptide having insecticidal activitycomprises an IP2-127 polypeptide.

21. A nucleic acid construct comprising the recombinant polynucleotideof any one of embodiments 1-20.

22. The nucleic acid construct of embodiment 21, further comprising apromoter operably linked to said recombinant polynucleotide.

23. A cell comprising at least one recombinant polynucleotide of any ofembodiments 1-20 or the nucleic acid construct of any one of embodiments21 or 22.

24. The cell of embodiment 23, wherein said cell is a plant cell.

25. The cell of embodiment 24, wherein said polynucleotide or nucleicacid construct is stably incorporated into the genome of said plantcell.

26. The cell of any one of embodiments 24 or 25, wherein said plant cellis from a monocot.

27. The cell of embodiment 26, wherein said monocot is maize, wheat,rice, barley, sorghum, sugarcane or rye.

28. The cell of any one of embodiments 24 or 25, wherein said plant cellis from a dicot.

29. The cell of embodiment 28, wherein the dicot is soybean, Brassica,sunflower, cotton or alfalfa.

30. A plant comprising at least one plant cell of any one of embodiments24-29.

31. A plant explant comprising at least one plant cell of any one ofembodiments 24-29.

32. A transgenic seed produced by the plant of embodiment 30, whereinsaid seed comprises said recombinant polynucleotide.

33. A recombinant polypeptide encoded by the polynucleotide of any oneof embodiments 1-20.

34. A method of targeting a polypeptide of interest to a chloroplastcomprising expressing the recombinant polynucleotide of any one ofembodiments 1-20 or the nucleic acid construct of embodiment 21 or 22 ina plant cell.

35. A method of targeting a polypeptide of interest to a chloroplastcomprising introducing the recombinant polynucleotide of any one ofembodiments 1-20 or the nucleic acid construct of embodiment 21 or 22 ina plant cell and expressing said recombinant polynucleotide in the plantcell.

36. The method of embodiment 34 or 35, wherein said method furthercomprises regenerating a transgenic plant from said plant cell.

37. The method of any one of embodiments 34-36, wherein said plant cellis from a monocot.

38. The method of embodiment 37, wherein said monocot is selected fromthe group consisting of maize, wheat, rice, barley, sorghum, sugarcaneor rye.

39. The method of any one of embodiments 34-36, wherein said plant cellis from a dicot.

40. The method of embodiment 39, wherein said dicot is selected from thegroup consisting of soybean, Brassica, sunflower, cotton or alfalfa.

41. The method of any one of embodiments 35-40, wherein said polypeptideof interest comprises an insecticidal protein and expression of saidpolypeptide controls a pest.

42. The method of embodiment 41, wherein said polypeptide of interestcomprises a Bacillus thuringiensis polypeptide having insecticidalactivity.

43. The method of embodiment 42, wherein said Bacillus thuringiensispolypeptide having insecticidal activity comprises an IP2-127polypeptide.

EXPERIMENTAL

The following examples are offered to illustrate, but not to limit, theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only, and persons skilledin the art will recognize various reagents or parameters that can bealtered without departing from the spirit of the invention or the scopeof the appended claims.

Example 1 Development of Novel Chloroplast Targeting Peptides (CTPs) forMaize

Nuclear encoded plant proteins that are translated in the cytosol aretargeted to the chloroplast using an N-terminal transit peptide. CTPsare both necessary and sufficient for correct chloroplast targeting andthese signal peptides are of variable length and sequence. Althoughthere is no consensus peptide sequence CTPs do share a similarstructural framework consisting of an uncharged N-terminus, a centralregion lacking acidic residues but enriched in hydroxylated amino acids,and a basic arginine-rich amphipathic C-terminus.

Two approaches were employed to develop these targeting peptides basedon the alignment of a set of known or predicted chloroplast transitpeptides from monocotyledonous species (see FIG. 1). The first approachutilized was to generate chimeric CTPs based on the predicted boundariesof the different CTP domains described above. The new CTPs can bederived from 3 or more different plant CTP sequences that when combinedtogether collectively reconstitute a CTP. A set of 5 different chimericCTPs were generated based on the CTP alignment found in FIG. 1. msCTP1(SEQ ID NO: 1: MALTTFSISRGGFVGALQGLKSTASLPNNESFSRHHLPSSSPQSSKRRCNLSFTTR) was generated from the combination of domains in sequential orderfrom Oryza sativa (Os) 1-deoxy-D xylulose-5-Phosphate Synthase CTP (aa1-17), Zea mays (Zm) ssRUBISCO CTP (aa 18-27) and Zm-beta-glucosidaseCTP (aa 28-56). msCTP2 (SEQ ID NO: 2:MGLSTVYSPAGPRLVPAPASLFQSPSSGCHSCWGPGPGGGRRLPS PRRRPITGTRS) was generatedfrom the combination of domains in sequential order from Zm-Malatedehydrogenase (NADP) CTP (aa 1-17), Os-Superoxide dismutase (SOD) CTP(aa 18-27) and Os-Soluble starch synthase CTP (aa 28-52). msCTP3 (SEQ IDNO: 3: MLSARAAATAAAAAASPPQPRLAATFLVLPSKRALAPLLSVGRVA TRRPRHVCQ) wasgenerated from the combination of the following domains in sequentialorder from Os-NADP-dependent Malic acid enzyme CTP (aa 1-17),Os-Phospho-2-dehydro-3-deoxyheptonate (PHD) Aldolase 2 CTP (aa 18-27),and Zm Thioredoxin M-type (TRX) CTP (aa 28-54). msCTP4 (SEQ ID NO: 4:MGLSTVYSPAAAAAASPPQPRSTASLPGCHSCWGPGPLLSVGRVATRRPR HVCQ) was generatedwith the combination of domains in sequential order from Zm-Malatedehydrogenase (NADP) CTP (aa 1-9), Os-NADP-dependent Malic acid enzymeCTP CTP (aa 10-21), Zm-ssRUBSICO CTP (aa 22-27), Os-Soluble starchsynthase CTP (aa 28-37), and Zm-Thioredoxin (TRX) CTP (aa 38-54). Thedesign of msCTP4 incorporated sequences derived from 2 separate CTPs forthe first and third domains. msCTP5 (SEQ ID NO: 5:MGLSTVYSPAAAAAASPPSLRSTASLPARPFHSLRLAAG RRGFACRGRSAAS) was generatedwith the combination of domains in sequential order from Zm-Malatedehydrogenase (NADP) CTP (aa 1-9), Os-NADP-dependent Malic acid enzymeCTP CTP (aa 10-17), Os-L-Ascorbate peroxidase 5(OsAPx05) (aa 18-21);Zm-ssRUBSICO CTP (aa 22-27), Os-Superoxide dismutase (OsSOD) CTP (aa28-39), and Os-Phosphoglucan water dikinase (OsPGDK) CTP (aa 40-52).

The second approach used the most frequent amino acid at each positionbased on the alignment of the different CTPs that were of a similar size(50-60 aa). In some cases where no dominant amino acid residue wasapparent one of the more frequent amino acid residues was chosen to beincorporated into the sequence. Two CTPs were developed using thisstrategy. msCTP6 (SEQ ID NO: 6: MALASVMAAAAASVVSFPAGRGSGGSSVLRSRALSLAGSRRSAAAVRRLAL) and msCTP7 (SEQ ID NO: 7:MAVATVLAAAALAAVSPPGLRSSLGFPVVRRSLPSAARGGSPAATRRCRAA).

A comparison of the amino acid identity levels for the different CTPsdeveloped using this strategy is found in Table 1. The homology betweenall the CTPs ranged from 16-64%.

TABLE 1 msCTP identity table. msCTP1 msCTP3 msCTP4 msCTP5 msCTP2 msCTP6msCTP7 msCTP1 16 26 28 16 20 22 msCTP3 64 38 21 38 36 msCTP4 59 46 31 36msCTP5 33 34 44 msCTP2 35 26 msCTP6 47 msCTP7

Example 2 Construction of Vectors for Testing the Ability of the NovelCTPs to Target an Insecticidal Toxin to the Chloroplast

A transient expression vector was generated to evaluate the ability ofthe novel CTPs to target an insecticidal toxin, IP2-127, to the maizechloroplast. This vector contained a fusion gene with IP2-127 at theN-terminus and AcGFP at the C terminus separated by a short linkersequence (SEQ ID NO: 8:ATGGGCAACAGCGTGCTCAACAGCGGACGCACCACCATCTGCGACGCCTACAACGTGGCCGCGCACGACCCGTTCAGCTTCCAGCACAAGAGCCTCGACACCGTGCAGCGCGAGTGGACCGAGTGGAAGAAGAACAACCACAGCCTCTACCTCGACCCGATCGTGGGCACCGTGGCCAGCTTCCTCCTCAAGAAGGTGGGCAGCCTCGTGGGCAAGCGCATCCTCAGCGAGCTGCGCAACCTCATCTTCCCGAGCGGCAGCACCAACCTCATGCAGGACATCCTCCGCGAGACCGAGCAGTTCCTCAACCAGCGCCTCGACACCGACACCCTCGCCAGGGTGAACGCCGAGCTGACCGGCCTCCAGGCCAACGTGGAGGAGTTCAACCGCCAGGTGGACAACTTCCTCAACCCGAACCGCAACGCCGTGCCGCTCAGCATCACCAGCAGCGTGAACACCATGCAGCAGCTCTTCCTCAACCGCCTCCCGCAGTTCCAGATGCAGGGCTACCAGCTCCTGCTCCTGCCGCTCTTCGCCCAGGCCGCCAACCTCCACCTCAGCTTCATCCGCGACGTGATCCTCAACGCCGACGAGTGGGGCATCAGCGCCGCCACCCTCCGCACCTACCGCGACTACCTCAAGAACTACACCCGCGACTACAGCAACTACTGCATCAACACCTACCAGAGCGCCTTCAAGGGCCTCAACACCCGCCTCCACGGCACCCTCGAGTTCCGCACCTACATGTTCCTCAACGTCTTCGAGTACGTGAGCATCTGGAGCCTCTTCAAGTACCAGAGCCTCCTCGTGAGCAGCGGCGCCAACCTCTACGCCAGCGGCAGCGGCCCGCAGCAGACCCAGAGCTTCACCAGCCAGGACTGGCCGTTCCTCTACAGCCTCTTCCAGGTGAACAGCAACTACGTGCTCAACGGCTTCAGCGGCGCCAGGCTCAGCAACACCTTCCCGAACATCGGCGGCCTCCCGGGCAGCACCACCACCCACGCCCTCCTCGCGGCCAGGGTGAACTACAGCGGCGGCATCAGCAGCGGCGACATCGGCGCCAGCCCGTTCAACCAGAACTTCAACTGCAGCACCTTCCTCCCGCCGCTCCTCACCCCGTTCGTGCGCAGCTGGCTCGATAGCGGCAGCGACCGCGAGGGCGTGGCCACCGTGACCAACTGGCAGACCGAGAGCTTCGAGACCACACTCGGGCTCAGGAGCGGCGCCTTCACCGCCCGCGGCAACAGCAACTACTTCCCGGACTACTTCATCCGGAACATCTCCGGCGTTCCGTTGGTGGTCCGTAACGAGGATCTCAGGAGGCCGCTGCACTACAACGAGATCCGCAACATCGCTTCGCCCAGCGGGACCCCAGGTGGAGCACGGGCCTACATGGTGTCCGTGCACAACCGGAAGAACAACATCCACGCGGTCCATGAGAACGGCAGCATGATCCACCTGGCTCCTAACGACTACACGGGGTTCACAATCTCTCCGATCCATGCTACTCAAGTCAACAACCAGACCAGGACGTTCATCTCGGAGAAGTTCGGCAACCAGGGAGACTCCTTGAGGTTCGAGCAGAACAACACAACTGCCCGCTACACCCTTCGGGGCAACGGGAACAGCTACAACCTCTACCTGCGCGTCAGCTCCATCGGCAACTCGACGATCAGGGTCACGATCAACGGAAGGGTCTACACTGCGACCAACGTGAACACGACAACTAACAACGACGGCGTCAACGACAACGGCGCTAGGTTCTCCGACATCAACATCGGGAACGTTGTGGCAAGCTCCAACTCGGATGTCCCTCTTGACATCAACGTCACCTTCAACTCTGGAACGCAGTTCGATCTGATGAACACAATGCTGGTGCCAACTAACATCAGCCCTCTGTACGGTGGAGGCGGCAGCGGTGGCGGAGGCTCCGGAGGCGGTGGCTCCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATCTTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTGAATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCCAAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCTGTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTACTTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATG AGCTGTACAAGTGA)which encoded a IP2-127-AcGFP fusion protein (SEQ ID NO: 9:MGNSVLNSGRTTICDAYNVAAHDPFSFQHKSLDTVQREWTEWKKNNHSLYLDPIVGTVASFLLKKVGSLVGKRILSELRNLIFPSGSTNLMQDILRETEQFLNQRLDTDTLARVNAELTGLQANVEEFNRQVDNFLNPNRNAVPLSITSSVNTMQQLFLNRLPQFQMQGYQLLLLPLFAQAANLHLSFIRDVILNADEWGISAATLRTYRDYLKNYTRDYSNYCINTYQSAFKGLNTRLHGTLEFRTYMFLNVFEYVSIWSLFKYQSLLVSSGANLYASGSGPQQTQSFTSQDWPFLYSLFQVNSNYVLNGFSGARLSNTFPNIGGLPGSTTTHALLAARVNYSGGISSGDIGASPFNQNFNCSTFLPPLLTPFVRSWLDSGSDREGVATVTNWQTESFETTLGLRSGAFTARGNSNYFPDYFIRNISGVPLVVRNEDLRRPLHYNEIRNIASPSGTPGGARAYMVSVHNRKNNIHAVHENGSMIHLAPNDYTGFTISPIHATQVNNQTRTFISEKFGNQGDSLRFEQNNTTARYTLRGNGNSYNLYLRVSSIGNSTIRVTINGRVYTATNVNTTINNDGVNDNGARFSDINIGNVVASSNSDVPLDINVTFNSGTQFDLMNTMLVPTNISPLYGGGGSGGGGSGGGGSMVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELYK) consisting of IP2-127 from amino acid 1-634, a short15 aa linker from amino acid 635-649, and AcGFP from amino acid 650-888.The IP2-127::AcGFP fusion gene is under control of the strongconstitutive maize Ubiquitin 1 promoter-5′UTR-intron1 regulatory elementin vector pSK-UB1-IP2-127::AcGFP with a pinII transcriptional terminatorsequence. The vector contains unique BamHI and KpnI restriction enzymesites immediately upstream of the IP2-127 translational start codon tofacilitate an in frame insertion of different CTP sequences at theN-terminus of the fusion.

The different novel monocot CTPs were synthesized by DNA2.0 (Menlo park,CA). Each CTP was subcloned into pSK-UB1-IP2-127::AcGFP using the uniqueBamHI and KpnI restriction sites. The base vector,pSK-UBI-IP2-127::AcGFP, was used as a control for non-CTP targetedIP2-127::AcGFP. A vector containing IP2-127::AcGFP fused to a previouslycharacterized CTP derived by gene shuffling (6H1-CTP) (SEQ ID NO: 10:MAATTLTSALPGAFSSSQRPSAPFNLQRSPRVLRRFNRKTGRQ PRGLVRAAKAQ) was used as apositive control for chloroplast targeting in transient expressionassays.

Example 3 Transient Expression Assays to Identify Novel CTPs Effectiveat Targeting IP2-127::AcGFP to the Maize Chloroplast

Maize seedlings were generated in soilless artificial condition byembedding kernels between two sheets of seed germination paper in a rolland its bottom portion was submerged in 0.1 mg/ml sucrose solution. Leafsegments were detached from seedlings at 15 days post-plantingimmediately before ballistic co-bombardment with colloidal goldparticles transformation. The lower epidermis of the leaf segments wereexcised and overlaid on top of filter papers in 100 mm Petri dishes.

The samples were co-bombarded with DNAs from both a DS-RED plasmidvector and individual CTP testing vectors using the PDS-1000 Hebiolistic particle delivery system (Bio-Rad, Hercules Calif.). Goldparticles (1.0 μm in diameter; Bio-Rad) were coated with plasmid DNAsfollowing the procedure described by Sanford et al. (1993) withmodifications. Briefly, 50 μl of freshly prepared gold particles inwater (20 mg/ml), and 20 μl of DNA mixture, which contain 10 μg ofequimolar quantities of the DS Red helper plasmid and CTP testingplasmids, were combined and 50 μl of a 2.5 M CaCl₂ solution and 20 μl offreshly prepared 0.1 M spermidine (Sigma-Aldrich, St Louis Mo.) wereslowly added with gentle vortexing. The mixture was incubated at roomtemperature for 5 min and pelleted at 13,000 g in a microcentrifuge for5 sec. The supernatant was carefully removed and the pellet wasresuspended in 85 μl of 100% ethanol. While gently vortexing, a 6 μlaliquot of suspension was drawn and dispensed onto the center of amacrocarrier membrane. The membrane was allowed to air dry completelyfor 2-5 min and used immediately. Leaf segments were bombarded at adistance of 9 cm from an 1100-psi rupture disk. Two replicate shots wereperformed from each coating preparation. After bombardment, the leafsamples were incubated in a moist chamber at 28 degree Celsius.

Initial examination was conducted at approximately 24 h post-bombardmentwith a Lumar fluorescence stereomicroscope (Carl Zeiss Inc., ThornwoodN.Y.) equipped with both a green-emitting (Zeiss Set 10) andred-emitting (Zeiss Set 43 HE) filter set to image the AcGFP and theDsRed2, respectively. The leaf segments containing AcGFP-positive cellsidentified in the stereomicroscope were placed in a 0.01% Tween 20solution and a vacuum was applied for about 10 min to remove internalair and to wet the leaf surface. The leaves were placed into coverglasschambers (Nalge Nunc International, Rochester N.Y.) in the samesolution, sealed with an additional coverglass and examined in theLSM510 (Carl Zeiss). AcGFP fluorescence was captured using a 488 nmargon laser for excitation and a 500-550 nm band pass emission filter.DsRed fluorescence was imaged using a 561 nm diode laser for excitationand a 575-615 nm band pass emission filter. Chlorophyll fluorescence wascaptured by combining 561 nm excitation and a 650-710 nm band passemission filter.

DsRed expression was used to assess the overall transformation rate andwas very useful for identifying transformed cells in the confocalmicroscope. Although epidermal cells were transformed with the highestfrequency by the bombardment procedure, mesophyll cells were used toassess plastid targeting. Plastid targeting was confirmed byco-localizing the AcGFP signal with chlorophyll fluorescence. Plastidtargeting was quantified with the confocal microscope by counting thenumber of mesophyll cells showing plastid-targeted AcGFP as a percentageof the total number of transformed cells (i.e., those exhibiting DsRedfluorescence).

The results of this analysis are outlined in Table 2. No colocalizationof IP2-127::AcGFP with the chloroplast was observed in the non-targetedcontrol where AcGFP fluorescence was limited exclusively to thecytosolic compartment. The majority of AcGFP derived fluorescence fromthe positive control, 6H1-CTP-IP2-127::AcGFP, was found to colocalize tochloroplasts and was scored at the highest level of +++. CTPs, msCTP1and msCTP4, showed equivalent levels of chloroplast colocalization ofIP2-127::AcGFP as observed with 6H1CTP. msCTP2 and msCTP6 directedIP2-127::AcGFP to the chloroplast although there was equal signalbetween chloroplast targeted and cytosolic localized fluorescenceobserved. This suggested that these two CTPs were not as efficient asmsCTP1 or msCTP4 in chloroplast targeting. msCTP5 directed morecytosolic localization of IP2-127::AcGFP than chloroplast colocalizationbut detectable levels of chloroplast colocalization was observed. msCTP7failed to direct any IP2-127::AcGFP to the chloroplast and was similarto the non-targeted control where IP2-127::AcGFP was cytosolic.

TABLE 2 Effectiveness of chloroplast targeting of novel CTPs based oncolocalization of AcGFP fluorescence with maize chloroplasts intransient expression assays. Colocalization with Construct chloroplastsIP2-127::AcGFP (non-targeted control) − 6H1CTP-IP2-127::AcGFP (targetedcontrol) +++ msCTP1-IP2-127::AcGFP +++ msCTP2-IP2-127::AcGFP ++msCTP3-IP2-127::AcGFP − msCTP4-IP2-127::AcGFP +++msCTP5-IP2-127::AcGFP + msCTP6-IP2-127::AcGFP ++ msCTP7-IP2-127::AcGFP −+++ IP2-127::AcGFP mostly chloroplast localized ++ equal IP2-127::GFPdetected in chloroplast and cytosol. + some IP2-127::AcGFP inchloroplast but mostly in cytosol − IP2-127::GFP entirely in cytosol

Example 4 Transgenic Plant Evaluation of Novel CTPs

The effect of chloroplast targeting was extended from transientexpression assays to stable transgenic events expressing IP2-127 withdifferent msCTPs. Chloroplast targeting of IP2-127 generally results inhigher accumulation of IP2-127 in plants than would be observed whennon-targeted. This difference in accumulation may be related to improvedstability of IP2-127 in the chloroplast and/or phytotoxicity issuesassociated with high levels of accumulation of IP2-127 in the cytosolduring the transformation process. Transformation vectors were generatedusing IP2-127 and a subset of the msCTPs-mCTP1, mCTP2, msCTP4, msCTP5,msCTP6—that were selected to represent the different qualitative resultsfrom the transient assays. Emphasis was put on those msCTPs thatdemonstrated some level of chloroplast colocalization in the transientexperiments. The transformation vectors were generated by using a basevector containing the IP2-127 gene with unique BamHI and KpnIrestriction enzyme sites directly upstream of the translation startcodon (ATG) of IP2-127. Subcloning each of the msCTPs into the BamHI andKpnI sites created an N-terminal fusion with the msCTP protein sequenceand the IP2-127 protein sequence. Non-targeted or msCTP targetedversions of the genes were placed under control of the maize Ubiquitin 1promoter-5′UTR-Ubiquitin intron1 and terminated with the pinIIterminator sequence from Potato creating the msCTP test cassette. ThemsCTP test cassettes were introduced into a binary transformation vectorusing standard Gateway™ LR Clonase reactions that were facilitated bythe presence of attL3 and attL4 recombination sites flanking the testcassette and attR3 and attL4 sites in the destination transformationvector. The final product of the LR reaction was a transformation binaryvector containing the msCTP-IP2-127 test cassette upstream of a cassettecontaining the maize Ubiquitin1 promoter-5′UTR-Ubiquitin intron1controlling expression of a PAT selectable marker gene with the 35Sterminator sequence.

Transgenic events derived from this set of msCTP testing vectors wereevaluated for expression of IP2-127 by ELISA. The results of the ELISAanalysis are shown in Table 3. Accumulation of IP2-127 in the cytosolwas 511 ppm. The addition of the 6H1-CTP to the N-terminus of IP2-127improved accumulation ˜2.5-fold to 1294 ppm demonstrating the effect ofan effective CTP on IP21-27 accumulation in plants. Two CTPs, msCTP1 andmsCTP4, improved IP2-127 accumulation ˜7.7-fold and ˜5-fold over thenon-targeted version and 2-3-fold over the level of accumulationdirected by 6H1-CTP. msCTP2 and msCTP6 showed comparable levels ofaccumulation as 6H1-CTP with both versions improving accumulation˜2.5-fold over non-targeted IP2-127. No significant improvement inIP2-127 accumulation was observed with msCTP5 as the levels ofaccumulation were only about ˜1.5-fold improved over the negativecontrol. Overall, accumulation of IP2-127 in transgenic plantscorrelated well with the results of colocalization of IP2-127::AcGFPobserved in transient expression assays. The results demonstrated thatthis strategy of developing new synthetic CTPs was effective atproviding novel chloroplast targeting peptides and that many of themsCTPs developed enhanced accumulation of IP2-127 in transgenic maizeplants.

TABLE 3 Accumulation of IP2-127 in transgenic maize events. IP2-127Expression Construct ID No of Events Tested (PPM) UBI-IP2-127 25 511UBI-6H1-CTP-IP2-127 21 1294 UBI-msCTP1-IP2-127 23 3931UBI-msCTP2-IP2-127 24 1408 UBI-msCTP4-IP2-127 25 2548 UBI-msCTP5-IP2-12723 802 UBI-msCTP6-IP2-127 25 1406

TABLE 4 Summary of CTP domains. CTP N-terminal Domain Central DomainC-terminal Domain msCTP1 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26msCTP2 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 msCTP3 SEQ ID NO: 30SEQ ID NO: 31 SEQ ID NO: 32 msCTP4 SEQ ID NO: 33/ SEQ ID NO: 35 SEQ IDNO: 36/ SEQ ID NO: 34 SEQ ID NO: 37 msCTP5 SEQ ID NO: 38/ SEQ ID NO: 40/SEQ ID NO: 42/ SEQ ID NO: 39 SEQ ID NO: 41 SEQ ID NO: 43

TABLE 5 Summary of SEQ ID NOS SEQ ID NO NA/AA Description 1 AA msCTP1 2AA msCTP2 3 AA msCTP3 4 AA msCTP4 5 AA msCTP5 6 AA msCTP6 7 AA msCTP7 8NA Nucleotide sequence of IP2-127-AcGFP fusion protein 9 AA Amino acidsequence of IP2-127-AcGFP fusion protein 10 AA 6H1-CTP (positive controlCTP) 11 AA CTP Consensus Sequence 12 AA IP2-127 Amino Acid sequence 13AA OS-1-deoxy-D-xyulose-5-Phosphate Synthase CTP 14 AA OS-Superoxidedismutase CTP 15 AA OS-soluble starch synthase CTP 16 AA OS-NADPdependent Malic acid enzyme CTP 17 AAOS-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 CTP 18 AAOS-L-Ascorbate Peroxidase 5 CTP 19 AA OS-Phosphoglucan water dikinase 20AA ZM-ssRUBISCO CTP 21 AA ZM-beta-glucosidase CTP 22 AA ZM-Malatedehydrogenase CTP 23 AA ZM-Thioredoxin M-type 24 AA Amino acids 1-17 ofOS-1-deoxy-D-xyulose-5-Phosphate Synthase CTP, N-terminal domain of CTP125 AA Amino acids 18-27 of ZM-ssRUBISCO CTP, Central domain of CTP1 26AA Amino acids 28-56 of ZM-beta-glucosidase CTP, C-terminal domain ofCTP1 27 AA Amino acids 1-17 of ZM-Malate dehydrogenase CTP, N- terminaldomain of CTP2 28 AA Amino acids 18-27 of OS-Superoxide dismutase CTP,Central domain of CTP2 29 AA Amino acids 28-52 of OS-soluble starchsynthase CTP, C- terminal domain of CTP2 30 AA Amino acids 1-17 ofOS-NADP dependent Malic acid enzyme CTP, N-terminal domain of CTP3 31 AAAmino acids 18-27 of OS-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2CTP, Central domain of CTP3 32 AA Amino acids 28-54 of ZM-ThioredoxinM-type, C-terminal domain of CTP3 33 AA Amino acids 1-9 of ZM-Malatedehydrogenase CTP, portion of N-terminal domain of CTP4 34 AA Aminoacids 10-21 of OS-NADP dependent Malic acid enzyme CTP, portion ofN-terminal domain of CTP4 35 AA Amino acids 22-27 of ZM-ssRUBISCO CTP,Central domain of CTP4 36 AA Amino acids 28-37 of OS-soluble starchsynthase CTP, portion of C-terminal domain of CTP4 37 AA Amino acids38-54 of ZM-Thioredoxin M-type, portion of C- terminal domain of CTP4 38AA Amino acids 1-9 of ZM-Malate dehydrogenase CTP, portion of N-terminaldomain of CTP5 39 AA Amino acids 10-17 of OS-NADP dependent Malic acidenzyme CTP, portion of N-terminal domain of CTP5 40 AA Amino acids 18-21of OS-L-Ascorbate Peroxidase 5 CTP, portion of central domain of CTP5 41AA Amino acids 22-27 of ZM-ssRUBISCO CTP, portion of central domain ofCTP5 42 AA Amino acids 28-39 of OS-Superoxide dismutase CTP, portion ofC-terminal domain of CTP5 43 AA Amino acids 40-52 of OS-Phosphoglucanwater dikinase, portion of C-terminal domain of CTP5

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A recombinant polynucleotide encoding achloroplast transit peptide (CTP) operably linked to a heterologouspolynucleotide encoding a polypeptide of interest, wherein the CTPcomprises a) an amino acid sequence comprising the amino acid sequenceset forth in SEQ ID NOS: 6 or 7; or b) an amino acid sequence having atleast 85% sequence identity to SEQ ID NOS: 6 or 7, wherein said aminoacid sequence has CTP activity.
 2. A recombinant polynucleotide encodinga chimeric CTP comprising a) an amino acid sequence comprising the aminoacid sequence set forth in SEQ ID NOS: 1,2 or 3; or b) an amino acidsequence having at least 85% sequence identity to SEQ ID NOS: 1, 2 or 3,wherein said amino acid sequence has CTP activity.
 3. A recombinantpolynucleotide encoding a chimeric CTP comprising a) an amino acidsequence comprising the amino acid sequence set forth in SEQ ID NO: 4;or b) an amino acid sequence having at least 85% sequence identity toSEQ ID NO: 4, wherein said amino acid sequence has CTP activity.
 4. Arecombinant polynucleotide encoding a chimeric CTP comprising a) anamino acid sequence comprising the amino acid sequence set forth in SEQID NO: 5; or b) an amino acid sequence having at least 85% sequenceidentity to SEQ ID NO: 5, wherein said amino acid sequence has CTPactivity.
 5. The recombinant polynucleotide of claim 1, wherein saidpolypeptide of interest comprises a Bacillus thuringiensis polypeptidehaving insecticidal activity.
 6. The recombinant polynucleotide of claim5, wherein said Bacillus thuringiensis polypeptide having insecticidalactivity comprises an IP2-127 polypeptide.
 7. A nucleic acid constructcomprising the recombinant polynucleotide of claim 1, 2, 3 or
 4. 8. Thenucleic acid construct of claim 7, further comprising a promoteroperably linked to said recombinant polynucleotide.
 9. A cell comprisingat least one recombinant polynucleotide of any one of claim 1, 2, 3 or4.
 10. The cell of claim 9, wherein said cell is a plant cell.
 11. Thecell of claim 10, wherein said polynucleotide is stably incorporatedinto the genome of said plant cell.
 12. The cell of claim 10, whereinsaid plant cell is from a monocot or dicot.
 13. The cell of claim 12,wherein said monocot is maize, wheat, rice, barley, sorghum, sugarcaneor rye, and wherein said dicot is soybean, Brassica, sunflower, cottonor alfalfa.
 14. A plant comprising at least one plant cell of claim 10.15. A plant explant comprising at least one plant cell of claim
 10. 16.A transgenic seed produced by the plant of claim 14, wherein said seedcomprises said recombinant polynucleotide.
 17. A recombinant polypeptideencoded by the polynucleotide of claim 1, 2, 3 or
 4. 18. A method oftargeting a polypeptide of interest to a chloroplast comprisingexpressing the recombinant polynucleotide of claim 1, 2, 3 or 4 in aplant cell.